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. 2021 Sep 9;11(19):9342–9357. doi: 10.7150/thno.62255

Figure 4.

Figure 4

The Gβγ-dependent AMPK/PKA pathway mediates the NmbR response. A, time course of IT changes induced by 100 nM Nmb in TG neurons dialyzed with QEHA (10 μM). Insets indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. B, summary data indicate the effect of 100 nM Nmb on IT in TG neurons dialyzed with QEHA (10 μM, n = 6) or SKEE (10 μM, n = 7). **p < 0.01 versus control, two-tailed t test. C, protein expression of Gβ in cells transduced with Gβ shRNA (Gβ-shRNA) or negative control shRNA (NC-shRNA). Blots depicted are representative of three independent experiments. #p < 0.05 versus NC-shRNA, two-tailed t test. D, summary data show the effect of Nmb (100 nM) on IT in TG neurons transduced with NC-shRNA (n = 9) or Gβ-shRNA (n = 8). *p < 0.05 versus NC-shRNA + control, one-way ANOVA followed by Bonferroni post hoc test. E, time course (left panel) and summary data (right panel) show the effect of Nmb (100 nM) on IT in cells pre-incubated with KT-5720 (1 μM, n = 7). Insets indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. **p < 0.01 versus control, two-tailed t test. F, exemplary current traces and summary of results show the effect of forskolin (10 µM) on IT in TG neurons in the absence (n = 7) or presence of KT-5720 (1 μM, n = 6). **p < 0.01 versus control, two-tailed t test. G, representative immunoblot and summary data show that treatment of TG cells with 100 nM Nmb did not affect the protein expression level of Cav3.2. GAPDH was used as the loading control. H, pretreatment of TG cells with 0.5 µM BIM23042, but not 1 μM KT-5720, attenuates the increased expression level of phosphorylated AMPK (p-AMPK) induced by 100 nM Nmb. GAPDH was used as the loading control. Blots depicted are representative of three independent experiments. *p < 0.05 versus control, two-tailed t test. I, summary data show the effect of 100 nM Nmb on PKA activity in TG cells pretreated with 0.5 µM BIM23042 or 10 µM compound C. All experiments were performed in triplicate with similar results. #p < 0.05 versus vehicle, one-way ANOVA followed by Bonferroni post hoc test. J, time course (left panel) and summary of results (right panel) indicate the effect of 100 nM Nmb on IT in TG neurons pretreated with compound C (10 µM, n = 7). Insets show the exemplary current traces. The Arabic numbers represent points used for representative traces. ***p < 0.001 versus control, two-tailed t test.